https://ash-bell.github.io/BIOM549-RNA-Seq-Tutorial/
Welcome to the BIOM549 bioinformatics workshop. The goal of this workshop is to provide you with some basic insight in how RNA-Seq data sets are analysed. Using this worksheet we will cover the following Intended Learning Outcomes (ILOs) for this module.
There are two sessions for this workshop both taught in person. It is not compulsory to attend but, these are the only times I am teaching. I would highly recommend you attend them.
| Date & Time | Location | Items |
|---|---|---|
| Wed 30 Nov 13:30 - 16:30 | Old Library 137 | Installing R and analysis walkthrough |
| Fri 16 Dec 10:30 - 13:00 | Amory B105 | How to present your data |
If you have any questions or queries outside of these times, you can email me at agb214@exeter.ac.uk and I will get back to you when I can.
In your first stream you should have either worked with zebrafish, plants or fungi. Each of those organisms would have been exposed to differing environmental conditions, which in turn, may result in them expressing different genes in response. Knowledge of which genes are expressed in different exposure conditions help us understand the host’s response to stimuli. Editing or monitoring these pathways can help increase organism fitness and act as bio-markers of disease. Measuring the difference in gene expression is the goal of RNA sequencing and is what we will be aiming to achieve in this tutorial.
Here you are provided with a data set from the plants stream where Arabidopsis thaliana pif4 mutants are grown at 22\(^\circ\)C and 27\(^\circ\)C with samples collected at 2 and 7 days exposure. Collected samples have their RNA extracted, sequenced and mapped back to their original genome to determine which genes are enriched. From there we get a “count” data set, representing the number of RNA sequences mapping back to each Arabidopsis gene. From here, we can devise several experimental questions.
You can get access to the data sets required for this workshop on the SharePoint link in the Data tab. You will need the count_data.csv, metadata.csv and Gene_annotations.tsv files.
These files will be available on Wed 30 Nov.
You are to provide a maximum of three plots that best answer the experimental questions set above for feedback in two weeks time - Tues 13 Dec. See the EXTRA: Better plotting examples for ideas to improve your plots.
During the Tues 13 Dec session I will go through your plots and help troubleshoot any issues. At the end of the second session you should have planned out and started on your poster due at the end of Term 2.
When you turn up to the sequencing practical, you need to bring your laptop. On your laptop you need to have R and RStudio installed before you can analyse any data. You can find instructions on how to do this below.
install.packages(c("tidyverse", "pheatmap", "ggupset", "ggridges", "europepmc")) 4. After tidyverse has
been installed (it will take a couple minutes), install the
BiocConductor packages the same way. Copy and paste the whole code chunk
into the console and press enter. This will take a couple of minutes.
After this, you’re done. You never need to re-install any package you
have previously installed.
if (!require("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install(c("DESeq2","clusterProfiler","enrichplot"))This portion starts the analysis. You should have access to three files on the SharePoint drive accessed through the BIOM549 VLE page. These are labelled metadata.csv, count_data.csv and Gene_annotations.tsv. The metadata table is a data frame containing which samples belong to which experimental group. The counts data frame is the output from the pre-processing analysis. This contains the number of reads that map to each Arabidopsis gene. The gene annotation file tell us the function of all genes within the Arabidopsis genome. Lets take a look at the data.
First we need create a script. This is like making a word document and records all the work you do. To make a script click File –> New File –> R Script. You can write and run anything here. Click on the save button and save this file somewhere sensible on your computer. Now download the metadata and counts file from SharePoint and save it in the same folder as your script. Everything we are going to do from now on will be done in this folder.
Next we need to tell the computer where is folder is located and
everything we do should be done here. To do that go to Session –> Set
Working Directory –> To Source File Location. A command will now run
in the console that sets your working directory setwd().
Copy and paste this command into your RScript, so that when you run this
script again, it always starts from the same place. Your script should
look something like this.
# Copy and paste your working directory here. This is mine and different to yours
setwd("C:/Users/agb214/OneDrive - University of Exeter/PhD/BIOM549/")If you recall we installed some R libraries previously. Although we never need to re-install our libraries, we need to tell R we want to use them each time we run our script (We never need to re-install Microsoft Word but we do need to tell windows to open it to use it). To do this, we load in our libraries one by one. Its good practice to include all your libraries at the start of your script so anyone else running your script knows what libraries they need to install to used your code. Copy and paste the following into your script. To run from your script, either click on the line you want to run and press Ctrl+Enter. This will only run the line that your cursor is on. To run multiple lines, highlight them all and press Ctrl+Enter. Do this now.
library(DESeq2)
library(tidyverse)
library(pheatmap)
library(clusterProfiler)
library(enrichplot)
library(ggupset)
library(ggridges)
library(europepmc)Now we can finally load in and look at our data specifying the first column of our data set is our row names. Again copy this into your script and run the code below.
count_data <- read.csv("count_data.csv", row.names = 1)
metadata <- read.csv("metadata.csv", row.names = 1)head(count_data)## Col.2d.22C.rep1 Col.2d.22C.rep2 Col.2d.22C.rep3 pif4.2d.22C.rep1
## AT1G01010 1225 1001 1256 1222
## AT1G01020 2076 1921 2035 2025
## AT1G01030 1115 1086 1102 1077
## AT1G01040 1733 1517 1795 1701
## AT1G01050 7031 7534 7171 7068
## AT1G01060 6946 8697 7634 7283
## pif4.2d.22C.rep2 pif4.2d.22C.rep3 Col.2d.27C.rep1 Col.2d.27C.rep2
## AT1G01010 1115 1341 1268 1190
## AT1G01020 1958 2075 1999 2248
## AT1G01030 1157 1107 863 815
## AT1G01040 1627 1634 1532 1657
## AT1G01050 6974 7050 7684 7627
## AT1G01060 7420 7216 5496 4936
## Col.2d.27C.rep3 pif4.2d.27C.rep1 pif4.2d.27C.rep2 pif4.2d.27C.rep3
## AT1G01010 1252 1198 1129 1318
## AT1G01020 2031 1854 2041 2440
## AT1G01030 919 832 803 908
## AT1G01040 1887 1868 1853 2069
## AT1G01050 7739 7086 7884 6818
## AT1G01060 7206 5438 5505 5767
## Col.7d.22C.rep1 Col.7d.22C.rep2 Col.7d.22C.rep3 pif4.7d.22C.rep1
## AT1G01010 1919 1908 2077 2263
## AT1G01020 2264 1898 1764 2214
## AT1G01030 806 672 641 1076
## AT1G01040 1873 1982 1968 2224
## AT1G01050 8393 9038 8774 7242
## AT1G01060 11844 13650 12584 10324
## pif4.7d.22C.rep2 pif4.7d.22C.rep3 Col.7d.27C.rep1 Col.7d.27C.rep2
## AT1G01010 2035 2012 2218 2387
## AT1G01020 2396 1695 2715 2863
## AT1G01030 1084 1054 638 800
## AT1G01040 2144 1884 1618 2177
## AT1G01050 7529 7433 8400 8003
## AT1G01060 10645 13128 9450 7605
## Col.7d.27C.rep3 pif4.7d.27C.rep1 pif4.7d.27C.rep2 pif4.7d.27C.rep3
## AT1G01010 2331 2311 2145 2202
## AT1G01020 2835 2949 2713 2924
## AT1G01030 761 1426 1092 1236
## AT1G01040 2032 2264 2080 2170
## AT1G01050 7651 7296 7932 7423
## AT1G01060 7344 8177 8648 7675
head(metadata)## Temperature Experimental_group Replicate Days_exposure
## Col.2d.22C.rep1 22 wildtype 1 2
## Col.2d.22C.rep2 22 wildtype 2 2
## Col.2d.22C.rep3 22 wildtype 3 2
## pif4.2d.22C.rep1 22 pif4_mutant 1 2
## pif4.2d.22C.rep2 22 pif4_mutant 2 2
## pif4.2d.22C.rep3 22 pif4_mutant 3 2
The command head() allows us to look at the first 5 rows
of our data. If we want to take a closer look we can instead use the
View() command which will open the data set in a new tab.
Beware using View() on very large data sets, you’ll freeze
your computer! For this exercise, you should be fine.
View(count_data)
View(metadata)As you can see, our count data set is the number of reads that mapped to each of the Arabidopsis genes for each of our samples. The metadata data set tells us which sample belongs to which exposure group. Pretty simple. Now lets see if any of the genes are significantly enriched (or not!) in our data set compare to the controls.
We need to combine our count data with our metadata. We use a package called DESeq2 which helps us keep track of multiple data sets in one experiment. Before we do that, we need to tell DESeq2 which is our control and experimental groups. To do this we set the column “Experimental group” as a factor, meaning they are categories and the first level (category) is “wildtype” or our control. Now we’re good to go.
metadata$Experimental_group <- as.factor(metadata$Experimental_group)
metadata$Experimental_group <- relevel(metadata$Experimental_group, ref = "wildtype")
metadata$Temperature <- as.factor(metadata$Temperature)
metadata$Replicate <- as.factor(metadata$Replicate)We also need to account that our experiment has multiple parameters,
not just two different experimental groups (pif4 and wildtype).
DESeq2 can account for this if you tell it. Here we define all our
groups, Temperature, Experimental group (wildtype or mutant) and Days
exposure (number of days exposed to each temperature).Here we also
include an interaction term: Days_exposure:Temperature
which tells us if the number of days exposed to each temperature
together have an compounded effect compared to if they were individually
measured.
ddsMF <- DESeqDataSetFromMatrix(countData = count_data,
colData = metadata,
design = ~Experimental_group)
design(ddsMF) <- formula(~ Temperature + Experimental_group + Days_exposure, Days_exposure:Temperature)
ddsMF## class: DESeqDataSet
## dim: 37336 24
## metadata(1): version
## assays(1): counts
## rownames(37336): AT1G01010 AT1G01020 ... ATMG09960 ATMG09980
## rowData names(0):
## colnames(24): Col.2d.22C.rep1 Col.2d.22C.rep2 ... pif4.7d.27C.rep2
## pif4.7d.27C.rep3
## colData names(4): Temperature Experimental_group Replicate
## Days_exposure
Notice when we type in ddsMF into our console, we now
have a DESeqDataSet. It has told us we have a metadata table and count
assay, with our row names as genes, columns as samples and metadata
categories as Temperature, Experimental_group, Replicate and
Days_exposure. Great!
We should remove genes that aren’t expressed frequently. This removes the “noise” which helps us find significant trends. These genes that are rarely expressed need to be in higher quantities so we can be sure they are significantly expressed, so we remove them. Here we remove any gene count that isn’t at least 1000 in X samples.
# Get the length of the number of columns in our "count_data" data set
X <- length(colnames(count_data))
# Subset our DESeq2 object where the minimum gene count across our entire data set is over 1000 per gene
keep <- rowSums(counts(ddsMF) >= 1000) >= X
ddsMF <- ddsMF[keep, ]The great thing about DDSeq2 is it does all our calculations for us. There are genes in our data set. Imagine manually filtering each one out! This is the power of using a coding language to analyse large data sets.
Now we can calculate if the genes are statistically differential
expressed. We use the p.adjusted value to account for
multiple retesting - the idea that if you test for everything, by random
chance you will find something that is significant but its just a
“lucky” pattern. If you’re interested, you can find out more here.
ddsMF <- DESeq(ddsMF)
# Define our p-value (alpha) cutoff as 0.05
resMF <- results(ddsMF, alpha = 0.05)
# Give us a quick summary of the results
# LFC > 0 (up) is number of up regulated genes
# LFC < 0 (down) is the number of down regulated genes
summary(resMF)##
## out of 11593 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 4457, 38%
## LFC < 0 (down) : 4171, 36%
## outliers [1] : 0, 0%
## low counts [2] : 0, 0%
## (mean count < 1195)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
Over half of the genes are up-/down-regulated. However, its expected that at different temperatures and times gene expression will be different. If we just compare the experimental groups
resMF <- results(ddsMF, contrast = c("Experimental_group", "wildtype", "pif4_mutant"), alpha = 0.05)## [1] "Number of significantly up/down regulated genes: 680"
## [1] "Total number of genes in this dataset: 11593"
Now accounting for differences in temperature, repeats and days exposure, we have qa better picture and differences between the mutant and control.
So now we know there are a total number of genes that are significantly up/down regulated. But how does this look compared to the entire data set? We can plot this as a volcano plot.
plotMA(resMF, xlim=c(1000,20000), ylim=c(-0.5,0.5))
The dots in blue are significantly up/down regulated and the ones in
grey not. Lets take a closer look at the most significantly
differentially regulated gene.
plotCounts(ddsMF, gene=which.min(resMF$padj), intgroup="Experimental_group")
We can definitely see this gene is down-regulated in the pif4
mutant.
Its great that we can see genes that are differentially regulated, but what if we want to see more then one? We could plot multiple dot/boxplot like above, but that gets more difficult to visualise the more genes you have. An alternative is a heat map which, plots multiple genes at once.
resSig <- resMF[resMF$padj < 0.05, ]
norm_counts <- counts(ddsMF, normalized = T) %>%
data.frame()
filtered <- ddsMF[rownames(ddsMF) %in% rownames(resSig), ]
pheatmap(assay(filtered), show_rownames = F)
We can definitely see differences in multiple genes now. Lets pick the
top 10 most signficantly differentially expressed genes.
resSig <- resMF[resMF$padj < 0.05, ]
resSig <- resSig[order(resSig$padj), ][1:10, ]
filtered <- ddsMF[rownames(ddsMF) %in% rownames(resSig), ]
pheatmap(assay(filtered))
This helps us identify by eye which genes are worth a closer look at.
There is a clear division between pif4 mutant and control experimental
groups.
Its great that we now know which genes are differentially expressed, but what do these genes do? We therefore need to determine the function of each gene in the Arabidopsis genome and we can then speculate if gene X is enriched in a certain treatment, it might result in Y. This is partly why we work on a model organsism such as Arabidopsis as there are hundreds if not thousands of studies that have already characterised the function of genes for us. Theses are then classified as genome ontology terms (GO terms) which give a curated name and function to each gene.This is because many studies may refer to a gene differently, so we standardised the names and functions in a database. I have already downloaded the gene name to Go term database for you, and you can find more about that here. Lets load in the database and take a look
annotations <- read.csv("Gene_annotations.tsv", sep = "\t")
head(annotations)## Locus TAIR.internal.locus.id Gene.Model.s.
## 1 AT1G01010 2200935 AT1G01010
## 2 AT1G01010 2200935 AT1G01010
## 3 AT1G01010 2200935 AT1G01010
## 4 AT1G01010 2200935 AT1G01010
## 5 AT1G01010 2200935 AT1G01010
## 6 AT1G01010 2200935 AT1G01010
## GO.term GO.ID
## 1 response to abscisic acid GO:0009737
## 2 response to water deprivation GO:0009414
## 3 cellular response to oxygen-containing compound GO:1901701
## 4 regulation of DNA-templated transcription GO:0006355
## 5 transcription cis-regulatory region binding GO:0000976
## 6 regulation of DNA-templated transcription GO:0006355
## TAIR.internal.GO.id category
## 1 11395 proc
## 2 5647 proc
## 3 44644 proc
## 4 7461 proc
## 5 35767 func
## 6 7461 proc
## GO.Slim.s.
## 1 response to chemical | response to endogenous stimulus
## 2 response to abiotic stimulus | response to stress | response to chemical
## 3 response to chemical | other cellular processes
## 4 nucleobase-containing compound metabolic process | other cellular processes | other metabolic processes | biosynthetic process
## 5 nucleic acid binding | DNA binding
## 6 nucleobase-containing compound metabolic process | other metabolic processes | other cellular processes | biosynthetic process
## Evidence.code Reference Made.by
## 1 IEA Publication:501796011|PMID:34562334
## 2 IEA Publication:501796011|PMID:34562334
## 3 IEA Publication:501796011|PMID:34562334
## 4 IEA AnalysisReference:501756966
## 5 IPI Publication:501786139|PMID:30356219
## 6 ISS Publication:1345963|PMID:11118137
## date.last.modified
## 1 10/15/2021 00:00:00
## 2 10/15/2021 00:00:00
## 3 10/15/2021 00:00:00
## 4 09/19/2022 00:00:00
## 5 12/18/2020 00:00:00
## 6 04/01/2021 00:00:00
We can see from this data set we have the gene names (Locus), Go.term (specific function), GO.ID (GO term), category (is this gene function : proc/bp/biological process, func/fc/molecular function or comp/cc/cellular process), Go.Slim.s (general function) and various other details on how the gene function was determined.
We want to know if our function (gene set) is differentially
expressed in our exposure group compared to our control. However, we
have differing numbers of genes that are part of the same function (for
example, we may have 10 genes that regulate height, but 100 genes
controlling growth rate). One gene that that is part of a small gene set
is more influential and changing the overall function of an organism
then one gene from a large gene set. Therefore, we need information into
the gene set size when determining if a function is differentially
expressed. This is calculated as a geneRatio. This is a complicated
equation and you can find out more here,
but basically it is the fraction of differentially expressed genes found
in the gene set. To do that, we use the GSEA function from
the library clusterProfiler and plot the results using
enrichplot.
The GSEA function requires three inputs, a list of genes
and the log2fold change we get from DESeq2’s
results function from our multifactor analysis, a term to
gene data frame containing our Go terms and gene names, and lastly a
term to names data frame with our Go terms to specific Go term
functions. We also need to correct for multiple retesting and we use the
Benjamini-Hochberg method. We also use an eps of 0 to allow
us to test for p-values smaller then the default 1e^-06.
FC <- resMF %>%
data.frame() %>%
select(log2FoldChange) %>%
rownames_to_column(var = "Locus")
geneList <- FC$log2FoldChange
names(geneList) <- as.character(FC$Locus)
geneList = sort(geneList, decreasing = TRUE)
T2G <- annotations %>%
select(GO.ID, Locus) %>%
unique()
T2N <- annotations %>%
select(GO.ID, GO.term) %>%
unique()
GSE <- GSEA(geneList = geneList, TERM2GENE = T2G, TERM2NAME = T2N, pAdjustMethod = "BH", pvalueCutoff = 0.05, seed = 1234, eps = 0)## preparing geneSet collections...
## GSEA analysis...
## leading edge analysis...
## done...
GSE## #
## # Gene Set Enrichment Analysis
## #
## #...@organism UNKNOWN
## #...@setType UNKNOWN
## #...@geneList Named num [1:11593] 1.046 0.939 0.86 0.848 0.748 ...
## - attr(*, "names")= chr [1:11593] "AT5G07010" "AT5G35940" "AT5G22500" "AT5G02540" ...
## #...nPerm
## #...pvalues adjusted by 'BH' with cutoff <0.05
## #...48 enriched terms found
## 'data.frame': 48 obs. of 11 variables:
## $ ID : chr "GO:0000325" "GO:0005829" "GO:0022626" "GO:0005773" ...
## $ Description : chr "plant-type vacuole" "cytosol" "cytosolic ribosome" "vacuole" ...
## $ setSize : int 198 402 51 105 211 58 488 102 62 38 ...
## $ enrichmentScore: num 0.484 0.385 0.709 0.536 0.413 ...
## $ NES : num 2.36 2.04 2.73 2.39 2.02 ...
## $ pvalue : num 1.58e-12 7.52e-12 2.65e-11 3.24e-09 2.11e-08 ...
## $ p.adjust : num 6.46e-10 1.53e-09 3.61e-09 3.31e-07 1.73e-06 ...
## $ qvalue : num 5.07e-10 1.20e-09 2.83e-09 2.59e-07 1.35e-06 ...
## $ rank : num 3230 2835 2595 2726 2696 ...
## $ leading_edge : chr "tags=57%, list=28%, signal=42%" "tags=43%, list=24%, signal=33%" "tags=78%, list=22%, signal=61%" "tags=53%, list=24%, signal=41%" ...
## $ core_enrichment: chr "AT1G22740/AT2G27730/AT1G19910/AT1G11260/AT1G20010/AT2G28900/AT1G04820/AT1G58030/AT1G78380/AT1G47128/AT1G04410/A"| __truncated__ "AT1G02340/AT1G12080/AT1G64660/AT2G23600/AT2G03680/AT1G62480/AT1G06850/AT2G29960/AT1G19350/AT1G20440/AT1G70830/A"| __truncated__ "AT1G01120/AT1G67090/AT1G03090/AT2G05840/AT1G20620/AT1G66200/AT2G27720/AT1G43170/AT1G07610/AT1G02780/AT1G67430/A"| __truncated__ "AT2G24280/AT2G23600/AT1G19910/AT2G24270/AT2G22170/AT1G75780/AT1G47128/AT1G11910/AT1G72150/AT2G29550/AT2G25450/A"| __truncated__ ...
## #...Citation
## T Wu, E Hu, S Xu, M Chen, P Guo, Z Dai, T Feng, L Zhou, W Tang, L Zhan, X Fu, S Liu, X Bo, and G Yu.
## clusterProfiler 4.0: A universal enrichment tool for interpreting omics data.
## The Innovation. 2021, 2(3):100141
Lets take a look at some of the plots.
upsetplot(GSE)
This is an upset plot showing you in each of the Go Terms, if it has
multiple functions (involved in more that one pathway), what its gene
ratio is.
dotplot(GSE, showCategory=10, split=".sign") +
facet_grid(.~.sign)
For the top ten gene sets which functions are enriched or suppressed,
how many Go terms are contribution to that gene set and is the
statistically significant.
ridgeplot(GSE, showCategory=10) +
labs(x = "Gene Ratio Distribution")## Picking joint bandwidth of 0.0137
For the top ten gene sets, show the Gene Ratio of each Go term and plot
them as distributions.
emapplot(pairwise_termsim(GSE), showCategory = 100)
Of the top 100 gene sets, which ones are related by function and are
they statistically significant.
cnetplot(GSE, categorySize="pvalue", foldChange=geneList, showCategory = 1)## Warning in cnetplot.enrichResult(x, ...): Use 'color.params = list(foldChange = your_value)' instead of 'foldChange'.
## The foldChange parameter will be removed in the next version.
## Scale for size is already present.
## Adding another scale for size, which will replace the existing scale.
Which genes are contributing to the top gene set and what is the
log2fold change for each gene
pmcplot(GSE$Description[1:5], 2010:2018, proportion=FALSE)
Of the top 5 gene sets, between 2010 and 2018, how many studies on PMC
mentioned this gene set by name.
library(ggrepel)
vol_plot_data <- resMF %>%
data.frame() %>%
mutate(sig = padj < 0.05 & (log2FoldChange > 0.5 | log2FoldChange < -0.5),
label = ifelse(sig == "TRUE", row.names(.), ""))
ggplot(vol_plot_data, aes(x = log2FoldChange, y = -log10(padj))) +
geom_point(aes(colour = sig)) +
xlab("log2 fold change") +
ylab("-log10 adjusted p-value") +
theme_bw() +
theme(legend.position = "none") +
geom_text_repel(aes(label = label), box.padding = 0.5) +
geom_vline(xintercept = c(-0.5, 0.5), linetype="dotted") +
geom_hline(yintercept = -log10(0.05), linetype="dotted")
## Boxplot gene of interest
#Select your chosen gene
df <- plotCounts(ddsMF, gene=which.min(resMF$padj),
intgroup=c("Experimental_group", "Temperature"),
returnData = T) %>%
mutate(Temperature = as.factor(Temperature))
df$Temperature <- relevel(df$Temperature, ref = "27")
ggplot(df, aes(x = Experimental_group, y = count, fill = Temperature)) +
geom_boxplot() +
geom_dotplot(binaxis = 'y', stackdir='center', dotsize = 0.75,
position=position_dodge(0.75))## Bin width defaults to 1/30 of the range of the data. Pick better value with
## `binwidth`.
dotplot(GSE, showCategory=10, split=".sign") +
facet_grid(.~.sign) +
scale_color_gradient(high = "#56B1F7", low = "#132B43")## Scale for colour is already present.
## Adding another scale for colour, which will replace the existing scale.
ridgeplot(GSE, showCategory=10) +
labs(x = "Gene Ratio Distribution",
title = "Top 10 enriched functions") +
scale_fill_gradient(high = "#56B1F7", low = "#132B43")## Scale for fill is already present.
## Adding another scale for fill, which will replace the existing scale.
## Picking joint bandwidth of 0.0137
resSig <- resMF[resMF$padj < 0.05, ]
resSig <- resSig[order(resSig$padj), ][1:10, ]
filtered <- ddsMF[rownames(ddsMF) %in% rownames(resSig), ]
pheatmap(t(assay(filtered)),
color=colorRampPalette(c("blue1", "skyblue", "white", "pink", "red"))(50),
display_numbers = T, number_format = "%.0f",
annotation_row = metadata)